Nucleic acids that control seed and fruit development in plants

ABSTRACT

The invention provides methods of controlling endosperm development in plants.

FIELD OF THE INVENTION

The present invention is directed to plant genetic engineering. Inparticular, it relates to modulation of expression of genes controllingendosperm development in plants.

BACKGROUND OF THE INVENTION

A fundamental problem in biology is to understand how fertilizationinitiates reproductive development. In higher plants, the ovulegenerates the female gametophyte which is composed of egg, central,synergid and antipodal cells (Reiser, et al., Plant Cell, 1291-1301(1993)). All are haploid except the central cell which contains twodaughter nuclei that fuse prior to fertilization. One sperm nucleusfertilizes the egg to form the zygote, whereas another sperm nucleusfuses with the diploid central cell nucleus to form the triploidendosperm nucleus (van Went, et al., Embryology of Angiosperms, pp.273-318 (1984)). The two fertilization products undergo distinctpatterns of development. In Arabidopsis, the embryo passes through aseries of stages that have been defined morphologically as preglobular,globular, heart, cotyledon and maturation (Goldberg, R. B., et al.,Science (1994) 266: 605-614; Mansfield, S. G., et al., Arabidopsis: AnAtlas of Morphology and Development, pp. 367-383 (1994)). The primaryendosperm nucleus undergoes a series of mitotic divisions to producenuclei that migrate into the expanding central cell (Mansfield, S. G.,et al., Arab Inf Serv 27: 53-64 (1990); Webb, M. C., et al., Planta 184:187-195 (1991)). Cytokinesis sequesters endosperm cytoplasm and nucleiinto discrete cells (Mansfield, S. G., et al., Arab Inf Serv 27: 65-72(1990)) that produce storage proteins, starch, and lipids which supportembryo growth (Lopes, M. A. et al., Plant Cell 5: 1383-1399 (1993)).Fertilization also activates development of the integument cell layersof the ovule that become the seed coat, and induces the ovary to growand form the fruit, or silique, in Arabidopsis.

Control of the expression of genes that control egg and central celldifferentiation, or those that activate reproductive development inresponse to fertilization is useful in the production of plants with arange of desired traits. These and other advantages are provided by thepresent application.

SUMMARY OF THE INVENTION

The present invention provides methods of modulating fruit and seeddevelopment and other traits in plants. The methods involve providing aplant comprising a recombinant expression cassette containing an FIEnucleic acid linked to a plant promoter.

In some embodiments, transcription of the FIE nucleic acid inhibitsexpression of an endogenous FIE gene or activity the encoded protein.This embodiment is particularly useful, for instance, making embryo-lessseed and parthenocarpic fruit. Alternatively, expression of the FIEnucleic acid may enhance expression of an endogenous FIE gene or FIEactivity

In the expression cassettes, the plant promoter may be a constitutivepromoter, for example, the CaMV 35S promoter. Alternatively, thepromoter may be a tissue-specific promoter. Examples of tissue specificexpression useful in the invention include ovule-specific orembryo-specific expression. For instance, the promoter sequence from theFIE genes disclosed here can be used to direct expression in relevantplant tissues.

The invention also provides seed or fruit produced by the methodsdescribed above. The seed or fruit of the invention comprise arecombinant expression cassette containing an FIE nucleic acid.

DEFINITIONS

The phrase “nucleic acid sequence” refers to a single or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. It includes chromosomal DNA, self-replicating plasmids,infectious polymers of DNA or RNA and DNA or RNA that performs aprimarily structural role.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of an operably linked nucleic acid. As usedherein, a “plant promoter” is a promoter that functions in plants.Promoters include necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

The term “plant” includes whole plants, plant organs (e.g., leaves,stems, flowers, roots, etc.), seeds and plant cells and progeny of same.The class of plants which can be used in the method of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including angiosperms (monocotyledonous anddicotyledonous plants), as well as gymnosperms. It includes plants of avariety of ploidy levels, including polyploid, diploid, haploid andhemizygous.

A polynucleotide sequence is “heterologous to” an organism or a secondpolynucleotide sequence if it originates from a foreign species, or, iffrom the same species, is modified from its original form. For example,a promoter operably linked to a heterologous coding sequence refers to acoding sequence from a species different from that from which thepromoter was derived, or, if from the same species, a coding sequencewhich is different from any naturally occurring allelic variants.

A polynucleotide “exogenous to” an individual plant is a polynucleotidewhich is introduced into the plant by any means other than by a sexualcross. Examples of means by which this can be accomplished are describedbelow, and include Agrobacterium-mediated transformation, biolisticmethods, electroporation, and the like. Such a plant containing theexogenous nucleic acid is referred to here as an R₁ generationtransgenic plant. Transgenic plants which arise from sexual cross or byselfing are descendants of such a plant.

A “FIE nucleic acid” or “FIE polynucleotide sequence” of the inventionis a subsequence or full length polynucleotide sequence of a gene whichencodes a polypeptide involved in control of reproductive developmentand which, when mutated, allows for aspects of fertilization independentreproductive development. In some embodiments, the polypeptides of theinvention have substantial sequence identity (as defined below) to apolycomb group gene of Drosophila. An exemplary nucleic acid of theinvention is the Arabidopsis FIE1 and FIE3 sequences disclosed below.FIE polynucleotides are defined by their ability to hybridize underdefined conditions to the exemplified nucleic acids or PCR productsderived from them. An FIE polynucleotide is typically at least about30-40 nucleotides to about 3000, usually less than about 5000nucleotides in length. The nucleic acids contain coding sequence of fromabout 100 to about 2000 nucleotides, often from about 500 to about 1700nucleotides in length.

FIE nucleic acids are a new class of plant regulatory genes that encodepolypeptides with sequence identity to members of the polycomb groupgenes first identified in Drosophila. Polycomb group gene products andtheir homologues in other species are responsible for repression ofhomeotic genes. The proteins are a heterogenous group that interact witheach other to form large complexes that bind DNA and thereby controlgene expression. For a review of the current understanding of polycombcomplex genes see, Pirrotta Cur. Op. Genet. Dev. 7:249-258 (1997). Ninegroups of polycomb genes have been identified. FIE1 (SEQ ID NO: 1) isrelated to the group of polycomb genes encoding protein comprising a SETdomain (see, e.g., Jenuwein et al. Cell. Mol. Life Sci. 54:80-93 (1998).FIE3 (SEQ ID NO:3) is related to the group encoding proteins comprisingWD40 repeats (see, Gutjahr et al. EMBO J. 14:4296-4306 (1995).

In the case of both expression of transgenes and inhibition ofendogenous genes (e.g., by antisense, or sense suppression) one of skillwill recognize that the inserted polynucleotide sequence need not beidentical, but may be only “substantially identical” to a sequence ofthe gene from which it was derived. As explained below, thesesubstantially identical variants are specifically covered by the termFIE nucleic acid.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the terms “FIE nucleic acid”. In addition, theterm specifically includes those sequences substantially identical(determined as described below) with an FIE polynucleotide sequencedisclosed here and that encode polypeptides that are either mutants ofwild type FIE polypeptides or retain the function of the FIE polypeptide(e.g., resulting from conservative substitutions of amino acids in theFIE polypeptide). In addition, variants can be those that encodedominant negative mutants as described below.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The terms “identical” or percent “identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Whenpercentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to sequences or subsequences that have atleast 60%, preferably 80%, most preferably 90-95% nucleotide or aminoacid residue identity when aligned for maximum correspondence over acomparison window as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence, which hassubstantial sequence or subsequence complementarity when the testsequence has substantial identity to a reference sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T, and X determine the sensitivity and speed of the alignment. TheBLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4,and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Serine (S), Threonine (T);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,    Creighton, Proteins (1984)).

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid. Thus,a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two molecules or theircomplements hybridize to each other under stringent conditions, asdescribed below.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, highly stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength pH. Low stringency conditions are generallyselected to be about 15-30° C. below the T_(m). The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 time background hybridization.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cased, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.

In the present invention, genomic DNA or cDNA comprising FIE nucleicacids of the invention can be identified in standard Southern blotsunder stringent conditions using the nucleic acid sequences disclosedhere. For the purposes of this disclosure, suitable stringent conditionsfor such hybridizations are those which include a hybridization in abuffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and at least onewash in 0.2×SSC at a temperature of at least about 50° C., usually about55° C. to about 60° C., for 20 minutes, or equivalent conditions. Apositive hybridization is at least twice background. Those of ordinaryskill will readily recognize that alternative hybridization and washconditions can be utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantiallyidentical is if the reference sequence, amplified by a pair ofoligonucleotide primers, can then be used as a probe under stringenthybridization conditions to isolate the test sequence from a cDNA orgenomic library, or to identify the test sequence in, e.g., a northernor Southern blot.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the genetic map used to clone the FIE3 gene.

FIG. 2 shows the analysis of the sequence in the DNA shown in FIG. 1using the GENSCANW program.

FIG. 3 shows the position of primers used to PCR amplify sequences fromthe FIE3 gene region.

FIG. 4 shows the genetic map used to clone the FIE1 gene.

FIG. 5 shows the results of complementation tests establishing that asingle gene (FIE1) was present on the complementing cosmid (6-22) thatwas not fully encoded on either of the non-complementing cosmids (2-9and 2-8).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides molecular strategies for controlling seed andfruit development.

Reproduction in higher plants is unique because it is initiated by twofertilization events in the haploid female gametophyte. One spermnucleus fertilizes the egg to form the embryo. A second sperm nucleusfertilizes the central cell to form the endosperm, a unique tissue thatsupports the growth of the embryo. Fertilization also activates maternaltissue differentiation, the ovule integuments form the seed coat and theovary forms the fruit.

The present invention is based, at least in part, on the discovery of aset of female-gametophytic mutations, termed fie(fertilization-independent endosperm), and the subsequent cloning of thegenes involved. Three mutants are disclosed here fie1, fie2, and fie3,which have been mapped to chromosomes 1, 2, and 3 of Arabidopsis,respectively. The fie mutations affect the central cell, allowing forreplication of the central cell nucleus and endosperm developmentwithout fertilization. FIE/fie seed coat and fruit undergofertilization-independent differentiation, showing that the fie femalegametophyte is the source of signals that activates sporophytic fruitand seed coat development. Generally, the mutant fie alleles are nottransmitted by the female gametophyte. Inheritance of a mutant fieallele (e.g., fie3) by the female gametophyte usually results in embryoabortion, even when the pollen bears the wild-type FIE allele. In thecase of fie 1 and fie2, however, transmission of the trait occurs inabout 1% of the progeny from the female gametophyte. In contrast, thefie 1, fie2, and fie3 mutant alleles are passed through the malegametophyte (i.e., pollen) in normal fashion.

The isolated sequences prepared as described herein, can be used in anumber of techniques, for example, to suppress or enhance endogenous FIEgene expression. Modulation of FIE gene expression or FIE activity inplants is particularly useful, for example, in producing embryo-lessseed, parthenocarpic fruit, or as part of a system to generate apomicticseed.

Isolation of FIE Nucleic Acids

Generally, the nomenclature and the laboratory procedures in recombinantDNA technology described below are those well known and commonlyemployed in the art. Standard techniques are used for cloning, DNA andRNA isolation, amplification and purification. Generally enzymaticreactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. These techniques and various other techniques aregenerally performed according to Sambrook et al., Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989).

The isolation of FIE nucleic acids may be accomplished by a number oftechniques. For instance, oligonucleotide probes based on the sequencesdisclosed here can be used to identify the desired gene in a cDNA orgenomic DNA library. To construct genomic libraries, large segments ofgenomic DNA are generated by random fragmentation, e.g. usingrestriction endonucleases, and are ligated with vector DNA to formconcatemers that can be packaged into the appropriate vector. To preparea cDNA library, mRNA is isolated from the desired organ, such as ovules,and a cDNA library which contains the FIE gene transcript is preparedfrom the mRNA. Alternatively, cDNA may be prepared from mRNA extractedfrom other tissues in which FIE genes or homologs are expressed.

The cDNA or genomic library can then be screened using a probe basedupon the sequence of a cloned FIE gene disclosed here. Probes may beused to hybridize with genomic DNA or cDNA sequences to isolatehomologous genes in the same or different plant species. Alternatively,antibodies raised against an FIE polypeptide can be used to screen anmRNA expression library.

Alternatively, the nucleic acids of interest can be amplified fromnucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology can be used to amplify thesequences of the FIE genes directly from genomic DNA, from cDNA, fromgenomic libraries or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clone nucleicacid sequences that code for proteins to be expressed, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes. For ageneral overview of PCR see PCR Protocols: A Guide to Methods andApplications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.),Academic Press, San Diego (1990).

Appropriate primers and probes for identifying FIE sequences from planttissues are generated from comparisons of the sequences provided herewith other polycomb group genes. For instance, FIE1 can be compared tothe other polycomb genes containing the SET domain, such as theArabidopsis curly leaf gene (Goodrich et al. Nature 386:44-51 (1997)) orthe Drosophila enhancer of zeste (E(z)) gene. FIE3 can be compared togenes containing WD40 repeats, such as the extra sex combs (esc) genefrom Drosophila. Using these techniques, one of skill can identifyconserved regions in the nucleic acids disclosed here to prepare theappropriate primer and probe sequences. Primers that specificallyhybridize to conserved regions in FIE1 or FIE3 genes can be used toamplify sequences from widely divergent plant species.

Standard nucleic acid hybridization techniques using the conditionsdisclosed above can then be used to identify full length cDNA or genomicclones.

Control of FIE Activity or Gene Expression

Since FIE genes are involved in controlling seed, in particularendosperm, development, inhibition of endogenous Fie activity or geneexpression is useful in a number of contexts. For instance, inhibitionof expression is useful in the development of parthenocarpic fruit(i.e., fruit formed in the absence of fertilization).

In addition, inhibition of FIE activity can be used for production offruit with small and/or degraded seed (referred to here as “seedlessfruit”) after fertilization. In many plants, particularly dicots, theendosperm is not persistent and eventually is degraded. Thus, in plantsof the invention in which Fie activity is inhibited, embryo-less seed donot persist and seedless fruit are produced.

Alternatively, plants of the invention can be used to preventpre-harvest sprouting in seeds, especially those derived from cereals.In these plants, the endosperm persists and is the major component ofthe mature seed. Premature growth of embryos in stored grain causesrelease of degradative enzymes which digest starch and other componentsof the endosperm. Plants of the present invention are useful inaddressing this problem because the seeds lack an embryo and thus willnot germinate.

In yet another use, nucleic acids of the invention can be used in thedevelopment of apomictic plant lines (i.e., plants in which asexualreproductive processes occur in the ovule, see, Koltunow, A. Plant Cell5: 1425-1437 (1993) for a discussion of apomixis). Apomixis provides anovel means to select and fix complex heterozygous genotypes that cannotbe easily maintained by traditional breeding. Thus, for instance, newhybrid lines with desired traits (e.g., hybrid vigor) can be obtainedand readily maintained.

One of skill will recognize that a number of methods can be used tomodulate FIE activity or gene expression. FIE activity can be modulatedin the plant cell at the gene, transcriptional, posttranscriptional,translational, or posttranslational, levels. Techniques for modulatingFIE activity at each of these levels are generally well known to one ofskill and are discussed briefly below.

Methods for introducing genetic mutations into plant genes are wellknown. For instance, seeds or other plant material can be treated with amutagenic chemical substance, according to standard techniques. Suchchemical substances include, but are not limited to, the following:diethyl sulfate, ethylene imine, ethyl methanesulfonate andN-nitroso-N-ethylurea. Alternatively, ionizing radiation from sourcessuch as, for example, X-rays or gamma rays can be used.

Alternatively, homologous recombination can be used to induce targetedgene disruptions by specifically deleting or altering the FIE gene invivo (see, generally, Grewal and Klar, Genetics 146: 1221-1238 (1997)and Xu et al., Genes Dev. 10: 2411-2422 (1996)). Homologousrecombination has been demonstrated in plants (Puchta et al.,Experientia 50: 277-284 (1994), Swoboda et al., EMBO J. 13: 484-489(1994); Offringa et al., Proc. Natl. Acad. Sci. USA 90: 7346-7350(1993); and Kempin et al. Nature 389:802-803 (1997)).

In applying homologous recombination technology to the genes of theinvention, mutations in selected portions of an FIE gene sequences(including 5′ upstream, 3′ downstream, and intragenic regions) such asthose disclosed here are made in vitro and then introduced into thedesired plant using standard techniques. Since the efficiency ofhomologous recombination is known to be dependent on the vectors used,use of dicistronic gene targeting vectors as described by Mountford etal. Proc. Natl. Acad. Sci. USA 91: 4303-4307 (1994); and Vaulont et al.Transgenic Res. 4: 247-255 (1995) are conveniently used to increase theefficiency of selecting for altered FIE gene expression in transgenicplants. The mutated gene will interact with the target wild-type gene insuch a way that homologous recombination and targeted replacement of thewild-type gene will occur in transgenic plant cells, resulting insuppression of FIE activity.

Alternatively, oligonucleotides composed of a contiguous stretch of RNAand DNA residues in a duplex conformation with double hairpin caps onthe ends can be used. The RNA/DNA sequence is designed to align with thesequence of the target FIE gene and to contain the desired nucleotidechange. Introduction of the chimeric oligonucleotide on anextrachromosomal T-DNA plasmid results in efficient and specific FIEgene conversion directed by chimeric molecules in a small number oftransformed plant cells. This method is described in Cole-Strauss et al.Science 273:1386-1389 (1996) and Yoon et al. Proc. Natl. Acad. Sci. USA93: 2071-2076 (1996).

Gene expression can be inactivated using recombinant DNA techniques bytransforming plant cells with constructs comprising transposons or T-DNAsequences. FIE mutants prepared by these methods are identifiedaccording to standard techniques. For instance, mutants can be detectedby PCR or by detecting the presence or absence of FIE mRNA, e.g., byNorthern blots. Mutants can also be selected by assaying for developmentof endosperm in the absence of fertilization.

The isolated nucleic acid sequences prepared as described herein, canalso be used in a number of techniques to control endogenous FIE geneexpression at various levels. Subsequences from the sequences disclosedhere can be used to control, transcription, RNA accumulation,translation, and the like.

A number of methods can be used to inhibit gene expression in plants.For instance, antisense technology can be conveniently used. Toaccomplish this, a nucleic acid segment from the desired gene is clonedand operably linked to a promoter such that the antisense strand of RNAwill be transcribed. The construct is then transformed into plants andthe antisense strand of RNA is produced. In plant cells, it has beensuggested that antisense suppression can act at all levels of generegulation including suppression of RNA translation (see, Bourque PlantSci. (Limerick) 105: 125-149 (1995); Pantopoulos In Progress in NucleicAcid Research and Molecular Biology, Vol. 48. Cohn, W. E. and K. Moldave(Ed.). Academic Press, Inc.: San Diego, Calif., USA; London, England,UK. p. 181-238; Heiser et al. Plant Sci. (Shannon) 127: 61-69 (1997))and by preventing the accumulation of mRNA which encodes the protein ofinterest, (see, Baulcombe Plant Mol. Bio. 32:79-88 (1996); Prins andGoldbach Arch. Virol. 141: 2259-2276 (1996); Metzlaff et al. Cell 88:845-854 (1997), Sheehy et al., Proc. Nat. Acad. Sci. USA, 85:8805-8809(1988), and Hiatt et al., U.S. Pat. No. 4,801,340).

The nucleic acid segment to be introduced generally will besubstantially identical to at least a portion of the endogenous FIE geneor genes to be repressed. The sequence, however, need not be perfectlyidentical to inhibit expression. The vectors of the present inventioncan be designed such that the inhibitory effect applies to other geneswithin a family of genes exhibiting homology or substantial homology tothe target gene.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about full length nucleotides should be used,though a sequence of at least about 100 nucleotides is preferred, asequence of at least about 200 nucleotides is more preferred, and asequence of about 500 to about 1700 nucleotides is especially preferred.

A number of gene regions can be targeted to suppress FIE geneexpression. The targets can include, for instance, the coding regions,introns, sequences from exon/intron junctions, 5′ or 3′ untranslatedregions, and the like. In some embodiments, the constructs can bedesigned to eliminate the ability of regulatory proteins to bind to FIEgene sequences that are required for its cell- and/or tissue-specificexpression. Such transcriptional regulatory sequences can be locatedeither 5′-, 3′-, or within the coding region of the gene and can beeither promote (positive regulatory element) or repress (negativeregulatory element) gene transcription. These sequences can beidentified using standard deletion analysis, well known to those ofskill in the art. Once the sequences are identified, an antisenseconstruct targeting these sequences is introduced into plants to controlgene transcription in particular tissue, for instance, in developingovules and/or seed.

Oligonucleotide-based triple-helix formation can be used to disrupt FIEgene expression. Triplex DNA can inhibit DNA transcription andreplication, generate site-specific mutations, cleave DNA, and inducehomologous recombination (see, e.g., Havre and Glazer J. Virology67:7324-7331 (1993); Scanlon et al. FASEB J. 9:1288-1296 (1995);Giovannangeli et al. Biochemistry 35:10539-10548 (1996); Chan and GlazerJ. Mol. Medicine (Berlin) 75: 267-282 (1997)). Triple helix DNAs can beused to target the same sequences identified for antisense regulation.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of FIE genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. Thus, ribozymes canbe used to target the same sequences identified for antisenseregulation.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs which arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Zhao and Pick Nature 365:448-451 (1993); Eastham andAhlering J. Urology 156:1186-1188 (1996); Sokol and Murray TransgenicRes. 5:363-371 (1996); Sun et al. Mol. Biotechnology 7:241-251 (1997);and Haseloff et al. Nature, 334:585-591 (1988).

Another method of suppression is sense cosuppression. Introduction ofnucleic acid configured in the sense orientation has been recently shownto be an effective means by which to block the transcription of targetgenes. For an example of the use of this method to modulate expressionof endogenous genes (see, Assaad et al. Plant Mol. Bio. 22: 1067-1085(1993); Flavell Proc. Natl. Acad. Sci. USA 91: 3490-3496 (1994); Stam etal. Annals Bot. 79: 3-12 (1997); Napoli et al., The Plant Cell 2:279-289(1990); and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184).

The suppressive effect may occur where the introduced sequence containsno coding sequence per se, but only intron or untranslated sequenceshomologous to sequences present in the primary transcript of theendogenous sequence. The introduced sequence generally will besubstantially identical to the endogenous sequence intended to berepressed. This minimal identity will typically be greater than about65%, but a higher identity might exert a more effective repression ofexpression of the endogenous sequences. Substantially greater identityof more than about 80% is preferred, though about 95% to absoluteidentity would be most preferred. As with antisense regulation, theeffect should apply to any other proteins within a similar family ofgenes exhibiting homology or substantial homology.

For sense suppression, the introduced sequence, needing less thanabsolute identity, also need not be full length, relative to either theprimary. transcription product or fully processed mRNA. This may bepreferred to avoid concurrent production of some plants which areoverexpressers. A higher identity in a shorter than full length sequencecompensates for a longer, less identical sequence. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andidentity of non-coding segments will be equally effective. Normally, asequence of the size ranges noted above for antisense regulation isused. In addition, the same gene regions noted for antisense regulationcan be targetted using cosuppression technologies.

Alternatively, FIE activity may be modulated by eliminating the proteinsthat are required for FIE cell-specific gene expression. Thus,expression of regulatory proteins and/or the sequences that control FIEgene expression can be modulated using the methods described here.

Another method is use of engineered tRNA suppression of FIE mRNAtranslation. This method involves the use of suppressor tRNAs totransactivate target genes containing premature stop codons (see,Betzner et al. Plant J. 11:587-595 (1997); and Choisne et al. Plant J.11: 597-604 (1997). A plant line containing a constitutively expressedFIE gene that contains an amber stop codon is first created. Multiplelines of plants, each containing tRNA suppressor gene constructs underthe direction of cell-type specific promoters are also generated. ThetRNA gene construct is then crossed into the FIE line to activate FIEactivity in a targeted manner. These tRNA suppressor lines could also beused to target the expression of any type of gene to the same cell ortissue types.

As noted above, FIE proteins as products of polycomb group genes arebelieved to form large complexes in vivo. Thus, production ofdominant-negative forms of FIE polypeptides that are defective in theirabilities to bind to other polycomb group proteins is a convenient meansto inhibit endogenous FIE activity. This approach involvestransformation of plants with constructs encoding mutant FIEpolypeptides that form defective complexes with endogenous polycombgroup proteins and thereby prevent the complex from forming properly.The mutant polypeptide may vary from the naturally occurring sequence atthe primary structure level by amino acid substitutions, additions,deletions, and the like. These modifications can be used in a number ofcombinations to produce the final modified protein chain. Use ofdominant negative mutants to inactivate target genes is described inMizukami et al. Plant Cell 8:831-845 (1996).

Another strategy to affect the ability of an FIE protein to interactwith itself or with other proteins involves the use of antibodiesspecific to FIE. In this method cell-specific expression of FIE-specificAbs is used inactivate functional domains through antibody:antigenrecognition (see, Hupp et al. Cell 83:237-245 (1995)).

Use of Nucleic Acids of the Invention to Enhance FIE Gene Expression

Isolated sequences prepared as described herein can also be used tointroduce expression of a particular FIE nucleic acid to enhance orincrease endogenous gene expression. For instance, polycomb genes areknown to control cell cycling. Enhanced expression can therefore be usedto control plant morphology by controlling whether or not cell divisiontakes place in desired tissues or cells. Enhanced expression can also beused, for instance, to increase vegetative growth by preventing theplant from setting seed. Where overexpression of a gene is desired, thedesired gene from a different species may be used to decrease potentialsense suppression effects.

One of skill will recognize that the polypeptides encoded by the genesof the invention, like other proteins, have different domains whichperform different functions. Thus, the gene sequences need not be fulllength, so long as the desired functional domain of the protein isexpressed.

Modified protein chains can also be readily designed utilizing variousrecombinant DNA techniques well known to those skilled in the art anddescribed in detail, below. For example, the chains can vary from thenaturally occurring sequence at the primary structure level by aminoacid substitutions, additions, deletions, and the like. Thesemodifications can be used in a number of combinations to produce thefinal modified protein chain.

Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of plant cells are prepared.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988). ADNA sequence coding for the desired polypeptide, for example a cDNAsequence encoding a full length protein, will preferably be combinedwith transcriptional and translational initiation regulatory sequenceswhich will direct the transcription of the sequence from the gene in theintended tissues of the transformed plant.

For example, for overexpression, a plant promoter fragment may beemployed which will direct expression of the gene in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumafaciens, and other transcription initiationregions from various plant genes known to those of skill. Such genesinclude for example, ACT11 from Arabidopsis (Huang et al. Plant Mol.Biol. 33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147,Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1from maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565(1989)), and Gpc2 from maize (GenBank No. U45855, Manjunath et al.,Plant Mol. Biol. 33:97-112 (1997)).

Alternatively, the plant promoter may direct expression of the FIEnucleic acid in a specific tissue or may be otherwise under more preciseenvironmental or developmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions, elevated temperature, or the presence of light.Such promoters are referred to here as “inducible” or “tissue-specific”promoters. One of skill will recognize that a tissue-specific promotermay drive expression of operably linked sequences in tissues other thanthe target tissue. Thus, as used herein a tissue-specific promoter isone that drives expression preferentially in the target tissue, but mayalso lead to some expression in other tissues as well.

Examples of promoters under developmental control include promoters thatinitiate transcription only (or primarily only) in certain tissues, suchas fruit, seeds, or flowers. Promoters that direct expression of nucleicacids in ovules, flowers or seeds are particularly useful in the presentinvention. As used herein a seed-specific promoter is one which directsexpression in seed tissues, such promoters may be, for example,ovule-specific (which includes promoters which direct expression inmaternal tissues or-the female gametophyte, such as egg cells or thecentral cell), embryo-specific, endosperm-specific, integument-specific,seed coat-specific, or some combination thereof. Examples include apromoter from the ovule-specific BEL1 gene described in Reiser et al.Cell 83:735-742 (1995) (GenBank No. U39944). Other suitable seedspecific promoters are derived from the following genes: MAC1 from maize(Sheridan et al. Genetics 142:1009-1020 (1996), Cat3 from maize (GenBankNo. L05934, Abler et al. Plant Mol. Biol. 22:10131-1038 (1993), the geneencoding oleosin 18 kD from maize (GenBank No. J05212, Lee et al. PlantMol. Biol. 26:1981-1987 (1994)), vivparous-1 from Arabidopsis (GenbankNo. U93215), the gene encoding oleosin from Arabidopsis (Genbank No.Z17657), Atmyc1 from Arabidopsis (Urao et al. Plant Mol. Biol.32:571-576 (1996), the 2 s seed storage protein gene family fromArabidopsis (Conceicao et al. Plant 5:493-505 (1994)) the gene encodingoleosin 20 kD from Brassica napus (GenBank No. M63985), napA fromBrassica napus (GenBank No. J02798, Josefsson et al. JBL 26:12196-1301(1987), the napin gene family from Brassica napus (Sjodahl et al. Planta197:264-271 (1995), the gene encoding the 2S storage protein fromBrassica napus (Dasgupta et al. Gene 133:301-302 (1993)), the genesencoding oleosin A (Genbank No. U09118) and oleosin B (Genbank No.U09119) from soybean and the gene encoding low molecular weight sulphurrich protein from soybean (Choi et al. Mol Gen, Genet. 246:266-268(1995)).

In addition, the promoter sequences from the FIE genes disclosed herecan be used to drive expression of the FIE polynucleotides of theinvention or heterologous sequences. The sequences of the promoters areidentified below.

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes of the invention will typically comprise a marker gene whichconfers a selectable phenotype on plant cells. For example, the markermay encode biocide resistance, particularly antibiotic resistance, suchas resistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or Basta.

Production of Transgenic Plants

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal. Embo J. 3:2717-2722 (1984). Electroporation techniques are describedin Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistictransformation techniques are described in Klein et al. Nature 327:70-73(1987).

Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. Agrobacterium tumefaciens-mediated transformation techniques,including disarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch et al. Science233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803(1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotypesuch as increased seed mass. Such regeneration techniques rely onmanipulation of certain phytohormones in a tissue culture growth medium,typically relying on a biocide and/or herbicide marker which has beenintroduced together with the desired nucleotide sequences. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp.124-176, MacMillilan Publishing Company, New York, 1983; and Binding,Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, BocaRaton, 1985. Regeneration can also be obtained from plant callus,explants, organs, or parts thereof. Such regeneration techniques aredescribed generally in Klee et al. Ann. Rev. of Plant Phys. 38:467486(1987).

The nucleic acids of the invention can be used to confer desired traitson essentially any plant. Thus, the invention has use over a broad rangeof plants, including species from the genera Anacardium, Arachis,Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria,Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus,Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana,Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea,Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum,Vicia, Vitis, Vigna, and Zea.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

Seed obtained from plants of the present invention can be analyzedaccording to well known procedures to identify plants with the desiredtrait. If antisense or other techniques are used to control Fie geneexpression, Northern blot analysis can be used to screen for desiredplants. In addition, the presence of fertilization independentreproductive development can be detected. Plants can be screened, forinstance, for the ability to form embryo-less seed, form seed that abortafter fertilization, or set fruit in the absence of fertilization. Theseprocedures will depend, part on the particular plant species being used,but will be carried out according to methods well known to those ofskill.

The following Examples are offered by way of illustration, notlimitation.

EXAMPLE 1

The following example describes methods used to identify the fiemutants. The methods described here are generally as described in Ohadet al., Proc. Natl. Acad. Sci. USA 93:5319-5324 (1996).

Materials and Methods

Growth and Phenotype of Plants.

Plants were grown under low humidity conditions (less than 50%) in glasshouses under 16 hr light/8 hr dark photoperiods generated bysupplemental lighting. Plants were grown at high humidity (greater than80%) in a lighted incubator (Percival, Boone, Iowa).

To test for fertilization-independent development, flower buds fromplants that had not yet begun to shed pollen (stage 12; (Smyth, D. R.,et al., Plant Cell 2:755-767 (1990))) were opened, immature anthers wereremoved, and the flower bud was covered with a plastic bag. Seven dayslater, the silique was measured, dissected, and the number of seed-likestructures and degenerating ovules were counted. To determine thefrequency of seed abortion following fertilization, siliques wereharvested 10 days after self-pollination, dissected, and wild-type andaborted seeds were counted.

Genetic Mapping.

Heterozygous FIE/fie (Landsberg erecta ecotype) plants were crossed asmales with female plants (Columbia ecotype). Because the mutant fieallele is only transmitted through the male gametophyte, FIE/fie progenywere crossed as males a second time to female gl1/gl1 (Columbia ecotype)plants. Approximately fifty-five progeny were scored for the segregationof the wild-type FIE and mutant fie alleles and for alleles of molecularmarkers as described previously (Bell, C., et al., Genomics 19:137-144(1994)). This analysis indicated that fie3 is located at approximatelyposition 30 on chromosome three, fie2 is located at approximatelyposition 65 on chromosome two, and fie1 is located at approximatelyposition 2 on chromosome one. Genetic recombination frequencies and mapdistances were calculated according to Koornneef and Stam (Koornneef,M., et al., Methods in Arabidopsis Research, pp. 83-99 (1992)) andKosambi (Kosambi, Ann. Eugen., 12: 172-175 (1944)).

Light Microscopy.

Nomarski photographs of whole-mount embryos and endosperm were obtainedby fixing longitudinally slit siliques in an ethanol:acetic acid (9:1)solution overnight, followed by two washes in 90% and 70% ethanol,respectively. Siliques were cleared with a chloralhydrate:glycerol:water solution (8:1:2, w:v:v) (Berleth, T., et al.,Devel 118: 575-587 (1993)). Whole mount preparations were fixed andstained with hematoxylin (Beeckman, T., et al., Plant Mol Biol Rep 12:37-42 (1994)). Embryo and endosperm were photographed with a ZeissAxioskop microscope (Carl Zeiss, Inc., Oberkochen, Germany) usingNomarski optics that permits visualization of optical sections withinthe seed.

GUS Histochemical Assays.

GUS activity was detected histochemically as described previously by(Beeckman, T., et al., Plant Mol Biol Rep 12: 37-42 (1994)).

Image Processing.

Photographs were scanned using a Microtek scanner. Pictures wereprocessed for publication using Adobe Photoshop 3.0 and printed on aTektronix Phaser 400 color printer.

Results

Isolation of Mutant Lines.

To begin to understand mechanisms that initiate reproductivedevelopment, we generated mutant Arabidopsis plants that undergo severalreproductive processes in the absence of fertilization. Arabidopsisplants homozygous for the conditional male sterile pop1 mutation(Preuss, D., et al., Genes and Devel 7: 974-985 (1993)) were used as theparental strain (Landsberg erecta ecotype). Fertility in pop1 plants issensitive to humidity because pop1 pollen do not hydrate properly due toa defect in wax biosynthesis. When grown at permissive condition, highrelative humidity (>80%), pop1 plants were male fertile and producedlong siliques with many viable seeds. By contrast, when grown atnon-permissive condition, low relative humidity (<50%), pop1 plants weremale sterile and produced short siliques with no seeds. Thus, siliqueelongation is a marker for reproductive events. To isolate mutations,homozygous pop1 seeds were mutagenized with ethylmethansulfonate (EMS)and approximately 50,000 M1 plants were screened for silique elongationat non-permissive conditions. Rare M1 plants were identified thatdisplayed heterozygous sectors with elongated siliques. These plantswere transferred to permissive conditions to insure the production ofviable M2 seed. Plants from M2 and M3 families grown at non-permissiveconditions were rechecked for non-sectored silique elongation. Toeliminate any effects of the pop1 mutation, or other EMS-induced lesionson the mutant phenotype, mutant plants were backrossed twice, as males,to wild-type plants. After removing the pop1 mutation,fertilization-independent phenotypes were confirmed after manual removalof anthers from immature flowers before pollen was shed. A total oftwelve lines were identified that displayed elongated siliques in theabsence of fertilization.

Fertilization-Independent Endosperm, Seed Coat and Silique Development.

In a representative line chosen for further study, heterozygous plantsproduced by back crosses to wild-type plants generated elongatedsiliques after anther removal with numerous seed-like structures. Theseresults indicated that heterozygous mutant plants were capable ofsilique elongation and seed-like structure development in the absence offertilization. We compared the development of the mutant seed-likestructures to that of wild-type seeds. After fertilization, theendosperm nucleus replicated and daughter nuclei migrated into theexpanding central cell. Ultimately, a syncytium of endosperm nuclei wasproduced. Nuclear divisions of the endosperm preceded the zygoticdivisions that formed the globular stage embryo. Embryo, endosperm orseed coat development did not occur in wild-type plants in the absenceof fertilization. Development of the ovule and female gametophyte inheterozygous mutant plants was normal. Just prior to flower opening,female gametophytes in these plants contained a single, prominentcentral cell nucleus. Subsequently, in the absence of fertilization,central cells with two large nuclei were detected. Further divisionsresulted in the production of additional nuclei that migrated into theexpanded central cell. Later in development, a nuclear syncytium wasformed with abundant endosperm nuclei. These results indicated that thecentral cell in mutant female gametophytes initiated endospermdevelopment in the absence of fertilization. We have named this mutationfie for fertilization-independent endosperm. By contrast, replication ofother nuclei in fie female gametophytes (egg, synergid, or antipodal)was not detected. Thus, the fie mutation specifically affectsreplication of the central cell nucleus.

We analyzed the frequency of multinucleate central cell formation in fiefemale gametophytes by comparing the percentage of multinucleate centralcells at three, five, and six days after emasculation of heterozygousFIE/fie and control wild-type flowers. At each time point, only 3% to 5%of wild-type central cells had more than one nucleus. Because none hadmore than two nuclei, most likely, these represented central cells withhaploid nuclei that had not fused during female gametophyte development.By contrast, the percentage of central cells in female gametophytes fromFIE/fie siliques with two or more nuclei increased from 21% to 47% overthe same time period. These results indicated that the fie mutationcaused a significant increase in formation of multinucleate centralcells in the absence of fertilization. The fact that close to 50% of thefemale gametophytes in heterozygous plants had multinucleate centralcells suggested that fie is a gametophytic mutation because a 1:1segregation of wild-type and mutant fie alleles occurs during meiosis.

We compared the fertilization-independent development of the maternalseed coat in FIE/fie seed-like structures to that of fertilizedwild-type seeds. The seed coat in wild-type Arabidopsis is generated bythe integuments of the ovule and surrounds the developing embryo andendosperm. Similarly, FIE/fie ovule integuments formed a seed coat thatsurrounded the developing mutant endosperm. These results indicated thatthe fie mutation activated both endosperm development and maternalsporophytic seed coat and silique differentiation that supportreproduction. No other effects on sporophytic growth and developmentwere detected in FIE/fie plants. The fie3 Mutant Allele Is NotTransmitted by the Female Gametophyte to the Next Generation.

To understand the mode of inheritance of the fie mutation, we analyzedthe progeny of reciprocal crosses. FIE3/fie3 females, crossed towild-type males, produced siliques with approximately equal numbers ofviable seeds with normal green embryos and nonviable white seeds withembryos aborted at the heart stage (344:375, 1:1, c2=1.3, P>0.2). Viableseeds from this cross were germinated and all 120 F1 progeny generatedwere wild-type. That is, none of the F1 progeny had significant levelsof F2 aborted seeds in their siliques after self-pollination. Nor didthe F1 progeny demonstrate fertilization-independent development. Thisindicated that presence of the fie mutant allele in the femalegametophyte, even when the male provided a wild-type allele, resulted inembryo abortion. Thus, the fie mutation is not transmitted by the femalegametophyte to the next generation. To study transmission of fie throughthe male gametophyte, we pollinated female wild-type plants with pollenfrom male FIE3/fie3 plants. Siliques from these crosses contained noaborted F1 seed. F1 plants were examined and a 1:1 segregation ofwild-type and FIE3/fie3 genotype was observed (62:58, c2=0.13, P>0.5).This indicated that wild-type and mutant fie3 alleles were transmittedby the male gametophyte with equal efficiency. That is, fie does notaffect male gametophyte, or pollen grain, function. Results fromreciprocal crosses were verified by analyzing the progeny fromself-pollinated FIE3/fie3 plants. Self-pollinated siliques displayed 1:1segregation of normal and aborted seeds (282:286, c2=0.03, P>0.8).Viable seed from self-pollinated siliques were germinated and a 1:1(71:64, c2=0.36, P>0.5) segregation of wild-type and FIE3/fie3 progenywas observed. These results confirmed that inheritance of a fie mutantallele by the female gametophyte resulted in embryo abortion, and thatinheritance of a fie mutant allele by the male gametophyte did notaffect pollen function. Thus, the wild-type FIE3 allele probably carriesout a function unique to the female gametophyte and does not appear tobe needed for male fertility.

In contrast, fie1 and fie2 mutant alleles were transmitted at lowfrequencies (about 1% of normal) through the female gametophyte. In thisway, fie1 homozygous mutants and fie2 homozygous mutants were obtainedthat appeared to display normal vegetative growth and development.

Discussion

In wild-type plants, fertilization initiates embryogenesis and endospermformation, and activates maternal seed coat and silique development. Theresults presented here indicate that specific aspects of plantreproductive development can occur in FIE/fie plants in the absence offertilization. These include silique elongation, seed coat formation,and endosperm development. Morphological analysis shows that earlyaspects of fertilization-independent fie endosperm development closelyresemble fertilized wild-type endosperm development. First, the fiecentral cell nucleus is stimulated to undergo replication. Second,nuclei that are produced migrate from the micropylar end of the centralcell and take up new positions in the central cell. Third, thedeveloping fie central cell expands to form an endosperm cavity. Thus,the requirement for fertilization to initiate these early events inendosperm formation has been eliminated by the fie mutation. Thissuggests that FIE plays a role in a signal transduction pathway thatlinks fertilization with the onset of central cell nuclear replicationand early endosperm development.

Mechanisms for Regulation of Endosperm Development by FIE.

One can envision two possible mechanisms for how FIE regulatesreplication of the central cell nucleus in response to fertilization.The protein encoded by the FIE gene may be involved in a positiveregulatory interaction. In this model, FIE is required for the centralcell to initiate endosperm development. Normally, fertilization isneeded for the presence of active FIE protein. The fie mutation resultsin the presence of active protein in the absence of fertilization.Alternatively, FIE may by involved in a negative regulatory interaction.In this model, the function of FIE protein is to prevent the centralcell from initiating endosperm development, and fertilization results inthe inactivation of FIE protein. The fie mutation results in theproduction of inactive protein, so that fertilization is no longerrequired to initiate endosperm development. However, complementationexperiments using transgenic plants indicate that FIE1 and FIE3 allelesare dominant over their respective mutant alleles. This indicates thatthe wild-type allele is involved in a negative regulatory interaction.Recently, it has been shown that cyclin-dependent kinase complexes,related to those that function in mammals, control the induction of DNAsynthesis and mitosis in maize endosperm (Grafi, G. et al., Science 269:1262-1264 (1995)). Because fie stimulates replication of the centralcell, fie may, either directly or indirectly, impinge upon cell cyclecontrol of the central cell nucleus, allowing replication to take placein the absence of fertilization.

Communication Between the Fie Female Gametophyte and the SporophyticOvule and Carpels.

The analysis of FIE/fie mutant plants has provided clues aboutinteractions between endosperm and maternal sporophytic tissues. FIE/fieovule integuments surrounding a mutant fie female gametophyte initiateseed coat development, whereas FIE/fie integuments in contact with aquiescent wild-type female gametophyte do not develop. This suggeststhat the FIE/fie ovule integuments initiate seed coat differentiation inresponse to a signal produced by the fie female gametophyte. We proposethat the source of the signal is the mutant fie central cell that hasinitiated endosperm development, although we cannot rule out theparticipation of other cells in the fie female gametophyte. In wild-typeplants, most likely, fertilization of the central cell produces anendosperm that activates seed coat development. This is consistent withexperiments showing that the maize endosperm interacts with nearbymaternal cells (Miller, M. E., et al., Plant Cell 4:297-305 (1992)).FIE/fie plants also display fertilization-independent elongation of theovary to form the silique. We propose that a signal is produced by thedeveloping seed-like structures to initiate silique elongation. This isin agreement with experiments suggesting that seeds are the source ofhormones, auxins and gibberellins, that activate fruit development (Lee,T. D. Plant Reproductive Ecology, pp. 179-202 (1988)). Taken together,these results suggest that the fertilized female gametophyte activatesmaternal developmental programs.

Relationship Between Fie and Apomixis.

Certain plant species display aspects of fertilization-independentreproductive development, including apomictic generation of embryo andendosperm, and development of the maternal seed coat and fruit (reviewedin (Koltunow, a. Plant Cell 5: 1425-1437 (1993)). The fie mutationreveals that Arabidopsis, a sexually reproducing plant, has the geneticpotential for aspects of fertilization-independent reproductivedevelopment. It is not known whether the mechanism offertilization-independent endosperm development conferred by the fiemutation is the same as autonomous endosperm formation observed incertain apomictic plant species. However, the fact that the fiephenotype is caused by a single genetic locus substantiates the viewthat the number of genetic differences between sexually and asexuallyreproducing plants is small (Koltunow, a. M., et al., Plant Physiol108:1345-1352 (1995)).

EXAMPLE 2

This example describes cloning of two Fie genes, Fie1 and Fie3.

Cloning The FIE3 Gene.

a. Mapping the position of the fie3 gene genetically. The fie3 mutationwas initially mapped to position 30 on chromosome 3, between AXR2 (auxinresistant dwarf) and EMB29 (embryo lethal). Next, two sets of F2 plantswith recombination breakpoints in the fie3 gene region were obtained.One set was between emb29 and fie3 and the other set was between axr2and fie3. As shown in FIG. 1A, these recombinants were used to map thefie3 gene relative to molecular markers (NDR, CH18, CH18S, BO20, AG20,KN1 and E13F12) that were obtained from overlapping YAC (yUP13F12), BAC(T1B4 and T4N1) and cosmid clones (FIG. 1A). YAC and BAC clones wereobtained from the Arabidopsis Stock Center (Ohio State University, USA).Cosmid subclones were generated in my laboratory. As shown in FIG. 1Aand 1B, this genetic analysis indicates that the fie3 gene resideswithin the 25 Kb region between the BO20 and AG20 markers.

b. Mapping the position of the fie3 gene by complementation experiments.To more precisely localize the fie3 gene, we analyzed a series ofoverlapping cosmid clones (BO20, GM15, AG20 and EI12) that span the fie3gene region. Each cosmid clone was tested for its ability to complementthe fie3 mutation in transgenic plants. Only cosmid GM 15complementedthe fie3 mutation (FIG. 1A). These results indicate that an essentialportion of the fie3 gene is in the 10 Kb region that is unique to cosmidGM 15. As shown in FIG. 1B, we have cloned DNA that spans this essentialportion of the fie3 gene and have determined its DNA sequence. As shownin FIG. 2, analysis of the sequence using the GENSCANW program revealeda gene with an open reading frame. The predicted cDNA sequence andpredicted amino acid sequence are shown in SEQ ID NO:3 and SEQ ID NO:4,respectively. Comparing the predicted amino acid sequence to those inpublic data bases revealed significant homology to the WD40 family ofPolycomb Group genes, and in particular, the “extra sex combs” gene inDrosophila. FIG. 3 shows the position of primers used to PCR amplifythis region. SEQ ID NO:5 provides the genomic DNA sequence of theWD40/Polycomb gene, plus approximately 3.8 Kb of 5′-flanking sequencesand 0.3 Kb of 3′-flanking sequences, plus the sequence of primers usedto PCR amplify this region. The transcription start site in SEQ ID NO:5is at position 3,872. Thus, the promoter sequence for FIE3 is locatedbetween position 1 and 3,872. The 5′-flanking and 3′-flanking regionscontain regulatory DNA sequences that control the expression of thisgene.

Cloning the FIE1 Gene.

a. Mapping the position of the FIE1 gene genetically. The fie1 mutationwas initially mapped to position 3 on chromosome 1, between AXR3 (auxinresistant dwarf) and EMB60 (embryo lethal). Next, two sets of F2 plantswith recombination breakpoints in the FIE1 gene region were obtained.One set was between emb60 and fie3 and the other set was between axr3and fie3. These recombinants were used to map the fie3 gene relative tomolecular markers (FIG. 4) that were obtained from an overlapping seriesof YAC and BAC clones from the Arabidopsis Stock Center (Ohio StateUniversity, USA).

b. Mapping the position of the FIE1 gene by complementation experiments.To more precisely localize the FIE1 gene, a series of overlapping cosmidclones (2-9, 6-22, 2-8) that span the FIE1 gene region were analyzed(FIG. 4). Each cosmid clone was tested for its ability to complement thefie1 mutation in transgenic plants. Only cosmid 6-22 complemented thefie1 mutation. The cosmids were analyzed for genes with open readingframes. FIG. 5 shows that a single gene was present on the complementingcosmid (6-22) that was not fully encoded on either of thenon-complementing cosmids (2-9 and 2-8). By RTPCR and 5′-race, the cDNAsequence of this gene and predicted amino acid of its protein wereobtained (SEQ ID NO: 1 and SEQ ID NO:2, respectively). Comparison of thepredicted amino acid sequence to those in public data bases revealedsignificant homology to the SET family of Polycomb Group Genes (e.g.,Enhancer of Zeste in Drosophila and Curly Leaf in Arabiopsis). Wecompared the wild-type and fie1 mutant sequence in 6-22. The onlydifference is a single base pair change that creates a prematuretranslation stop codon in the 5 ′-end of the set/polycomb group gene.The base pair change is at position 823 (C->T) on the cDNA sequenceshown in SEQ ID NO: 1.

SEQ ID NO:6 shows the genomic sequence of the FIE1 SET/polycomb gene,plus approximately 2 Kb of 5′-flanking sequences and approximately 0.7Kb of 3′-flanking sequences. The translation start site is located atposition 2036 of SEQ ID NO:6. Thus, the promoter sequence is locatedbetween position 1 and position 2036.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

1. An isolated nucleic acid molecule comprising a FIE polynucleotidesequence, which polynucleotide sequence specifically hybridizes to SEQID NO:1 or SEQ ID NO:3 under stringent conditions.
 2. The isolatednucleic acid molecule of claim I, wherein the FIE polynucleotide isbetween about at least about 100 nucleotides in length.
 3. The isolatednucleic acid molecule of claim 1, wherein the FIE polynucleotide is SEQID NO:1.
 4. The isolated nucleic acid molecule of claim 1, wherein theFIE polynucleotide is SEQ ID NO:3.
 5. The isolated nucleic acid moleculeof claim 1, further comprising a plant promoter operably linked to theFIE polynucleotide.
 6. The isolated nucleic acid molecule of claim 5,wherein the plant promoter is from a FIE1 gene.
 7. The isolated nucleicacid of claim 6, wherein the FIE polynucleotide is linked to thepromoter in an antisense orientation.
 8. An isolated nucleic acidmolecule comprising a FIE polynucleotide sequence, which polynucleotidesequence encodes FIE polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:4.9. a transgenic plant comprising an expression cassette containing aplant promoter operably linked to a heterologous FIE polynucleotide ofclaim
 1. 10. The transgenic plant of claim 9, wherein the heterologousFIE polynucleotide encodes a FIE polypeptide.
 11. The transgenic plantof claim 10, wherein the FIE polypeptide is as shown in SEQ ID NO:2 orSEQ ID NO:4.
 12. The transgenic plant of claim 9, wherein theheterologous FIE polynucleotide is linked to the promoter in anantisense orientation.
 13. The transgenic plant of claim 9, wherein theplant promoter is from a FIE gene.
 14. The transgenic plant of claim 13,wherein the FIE gene is as shown in SEQ ID NO:1 or SEQ ID NO:3.
 15. Amethod of modulating endosperm development in a plant, the methodcomprising introducing into the plant an expression cassette containinga plant promoter operably linked to a heterologous FIE polynucleotide.16. The method of claim 15, wherein the heterologous FIE polynucleotideencodes an FIE polypeptide.
 17. The method of claim 16, wherein the FIEpolypeptide has an amino acid sequence as shown in SEQ ID NO:2 or SEQ IDNO:4.
 18. The method of claim 15, wherein the heterologous FIEpolynucleotide is linked to the promoter in an antisense orientation.19. The method of claim 15, wherein the heterologous FIE polynucleotideis SEQ ID NO:1 or SEQ ID NO:3.
 20. The method of claim 15, wherein theplant promoter is from a FIE gene.
 21. The method of claim 15, whereinthe expression cassette is introduced into the plant through a sexualcross.